PROJECT SUMMARY: Rett syndrome is the most common genetic cause of intellectual disability in girls and is characterized by neurodevelopmental delay, abnormal arm movements, seizures, and autism spectrum behavior. It has been known for nearly a decade that Rett syndrome is caused by mutations in the MECP2 gene and that restoring normal levels of MECP2 in rodent models, even after symptom onset, can reverse most symptoms. While this genetic insight has provided hope for treatment, the incredible complexity of MeCP2 function in neurons has challenged the development of actionable therapeutic strategies. MeCP2 is known to be highly enriched in neurons and bind to methylated DNA, but its subtle effects on transcription have been difficult to understand and reconcile with the severity of Rett syndrome phenotypes. Our laboratory recently performed a meta-analysis of nearly a dozen independent studies of MeCP2-regulated genes and found that MeCP2 selectively controls the expression of very long genes. This observation is highly specific to MeCP2 and is observed in both rodent models and Rett syndrome patients. The high level of MeCP2 in neurons and the tendency for neuronal proteins to be large and encoded by long genes, may explain why MeCP2 mutations preferentially cause neuronal dysfunction. Our preliminary data supports a role of long gene misregulation in the pathogenesis of Rett syndrome because normalizing long gene expression directly with topoisomerase inhibitors improves Rett phenotypes in vitro and in vivo. In neurons, topoisomerases function to unwind DNA during transcription and are required for expression of long genes. The reciprocal control over long gene expression between MeCP2 and topoisomerase, as well as preliminary data demonstrating a physical interaction between these proteins, raise several important mechanistic and therapeutically relevant questions that are the focus of this proposal. 1) to characterize the cellular and behavioral effects of topoisomerase inhibition in the mouse model of Rett syndrome, and 2) to characterize the interaction between MeCP2 and topoisomerase. Together, these studies aim to inform a new therapeutic strategy for Rett syndrome focused on correcting long gene misregulation through direct control of topoisomerase activity.